Evasion of wheat resistance gene Lr15 recognition by the leaf rust fungus is attributed to the coincidence of natural mutations and deletion in AvrLr15 gene

Abstract Employing race‐specific resistance genes remains an effective strategy to protect wheat from leaf rust caused by Puccinia triticina (Pt) worldwide, while the newly emerged Pt races, owing to rapid genetic evolution, frequently overcome the immune response delivered by race‐specific resistance genes. The molecular mechanisms underlying the newly evolved virulence Pt pathogen remain unknown. Here, we identified an avirulence protein AvrLr15 from Pt that induced Lr15‐dependent immune responses. Heterologously produced AvrLr15 triggered pronounced cell death in Lr15‐isogenic wheat leaves. AvrLr15 contains a functional signal peptide, localized to the plant nucleus and cytosol and can suppress BAX‐induced cell death. Evasion of Lr15‐mediated resistance in wheat was associated with a deletion and point mutations of amino acids in AvrLr15 rather than AvrLr15 gene loss in the Lr15‐breaking Pt races, implying that AvrLr15 is required for the virulence function of Pt. Our findings identified the first molecular determinant of wheat race‐specific immunity and facilitated the identification of the first AVR/R gene pair in the Pt–wheat pathosystem, which will provide a molecular marker to monitor natural Pt populations and guide the deployment of Lr15‐resistant wheat cultivars in the field.

tified an avirulence protein AvrLr15 from Pt that induced Lr15-dependent immune responses.Heterologously produced AvrLr15 triggered pronounced cell death in Lr15-isogenic wheat leaves.AvrLr15 contains a functional signal peptide, localized to the plant nucleus and cytosol and can suppress BAX-induced cell death.Evasion of Lr15-mediated resistance in wheat was associated with a deletion and point mutations of amino acids in AvrLr15 rather than AvrLr15 gene loss in the Lr15-breaking Pt races, implying that AvrLr15 is required for the virulence function of Pt.Our findings identified the first molecular determinant of wheat race-specific immunity and facilitated the identification of the first AVR/R gene pair in the Pt-wheat pathosystem, which will provide a molecular marker to monitor natural Pt populations and guide the deployment of Lr15-resistant wheat cultivars in the field.

K E Y W O R D S
avirulence gene, AvrLr15, Puccinia triticina rapid emergence of new Pt isolates with the ability to avoid R gene recognition (Kolmer et al., 2007).Because the interaction between wheat and Pt follows the gene-for-gene model, the emergence of resistance-breaking Pt isolates highlights the urgent need to identify Pt avirulence genes and to decipher the molecular mechanisms underlying their recognition and evasion by R genes, which will guide and prioritize the deployment of resistance genes in different geographic regions worldwide.
A pathogen avirulence protein is perceived by its cognate immune receptor, which activates the host immune response that frequently results in localized programmed cell death (PCD) at the site of pathogen infection (Collins et al., 2011;Elmore et al., 2011).
To identify an avirulence protein of Pt, we selected a highly upregulated effector candidate gene PTTG_27353 (GenBank accession number: OAV93472.1)from our previous RNA-seq data (CNGBdb: PRJNA694214) derived from the susceptible wheat cv.Chinese Spring interacting with Pt race PHNT (Cui et al., 2023).To validate the gene expression detected in the RNA-seq data, the expression profiles of PTTG_27353 in Chinese Spring inoculated with Pt PHNT were examined by reverse transcription-quantitative PCR (RT-qPCR) (Text S1).In comparison with the expression level at 0 hour post-inoculation (hpi), the expression of PTTG_27353 was significantly induced and peaked at 12 hpi and subsequently decreased from 24 to 96 hpi (Figure 1a).Previous studies indicated that haustorial formation occurrs at 24 hpi (Prasad et al., 2020;Qi et al., 2018;Yang et al., 2020) suggesting that PTTG_27353 may be involved in the initial infection of wheat by Pt.Next, we produced the recombinant protein PTTG_27353 (Figure 1b, Table S1) using the Escherichia coli expression system (Figure 1c, Figure S1, Text S1) and infiltrated the purified protein into 42 near-isogenic lines of Thatcher carrying a single leaf rust R genes.Cell death occurred in the leaves of line TcLr15 (RL6052) when PTTG_27353 protein was infiltrated, but no cell death was evident in the leaves of the other 41 near-isogenic lines or in the negative control infiltrated with buffer (Figure S2).To confirm this initial observation, we repeated this experiment three times, and consistent results were obtained (Figure 1d).Therefore, from wheat fungal pathogens (Sung et al., 2021).For example, the infiltration of heterologously produced wheat stem rust effector protein AvrSr35 into Sr35 wheat leaves triggers strong cell death (Salcedo et al., 2017).In addition, infiltration of effector protein SnTox3 from Parastagonospora nodorum induces a strong necrotic response in susceptible wheat, while infiltration of TaPR1 proteins in wheat leaves can suppress P. nodorum disease (Sung et al., 2021).
To confirm the function of AvrLr15, we silenced the expression of AvrLr15 by BSMV-host-induced gene silencing (HIGS) in TcLr15 wheat (Text S1).Compared with the control plants, more urediniospores appeared on the leaves of AvrLr15-silenced plants after inoculation with the avirulent race PHNT (Figure 2).The silencing efficiency, monitored by RT-qPCR, indicated that the relative expression of AvrLr15 was significantly reduced by 76% and 72% after infection with avirulent Pt at 24 and 48 hpi, respectively (Figure S3).
All these results indicate that AvrLr15 triggers the disease resistance response of TcLr15.
The full-length AvrLr15 gene was 420 bp, including two exons and one intron (Figure S4).A primer pair in the untranslated region (UTR) was used to determine the introns and exons of the genomic DNA sequences of different races (Figures S5 and S6, Table S1).Rust fungi from different plant species were examined using PCR, and the results showed that AvrLr15 could only be detected in wheat leaf rust fungi (Figure S7).The coding sequence of AvrLr15 encodes a 13 kDa protein corresponding to 110 amino acids, without a known conserved domain and containing four cysteine residues.SignalP v. 5.0 predicted the presence of an N-terminal signal peptide containing 19 amino acids in the AvrLr15 protein, and effector P-Fungi v.
3.0 preded that AvrLr15 may be an effector that could be secreted into the host (Figure S8).To examine the functional role of the putative signal peptide (SP) of AvrLr15, the yeast signal sequence trap system (Jacobs et al., 1997) was employed (Text S1).SP AvrLr15 was cloned and ligated to the pSUC2 vector (Figure S9, Table S1).
Yeast strains YTK12 transformed with SP AvrLr15 or with a construct with the signal peptide of Ps87 (as positive control; Gu et al., 2011) were able to grow on CMD − W and YPRAA media (Figure 3a).2,3,5-triphenyltetrazolium chloride (TTC)-treated Ps87 and SP AvrLr15 culture filtrates turned red, whereas the negative control culture filtrates treated with TTC remained colourless (Figure 3a).These results indicated that AvrLr15 does contain a functional signal peptide responsible for the secretion of AvrLr15 into the plant cell.Inhibition of BAX-induced cell death is often regarded as a criterion for suppressing plant immunity (Tao et al., 2021) (Text S1).ΔSP AvrLr15 was cloned and ligated to the pCamA vector (Figure S10, Table S1).To examine the ability of AvrLr15 to inhibit BAX-induced cell death, we infiltrated Agrobacterium tumefaciens carrying pCamA: ΔSP Av-rLr15 into Nicotiana benthamiana and 24 hours later A. tumefaciens carrying pEarlyGate100:BAX was infiltrated.Figure 1f shows that ΔSP AvrLr15 completely inhibited BAX-induced cell death in N. benthamiana (Figure 3b).The expression of GFP-tagged ΔSP AvrLr15 in pCamA: ΔSP AvrLr15 after agroinfiltration in N. benthamiana leaves was confirmed by green fluorescent signals under microscopy; ΔSP AvrLr15 localized in the plant nucleus and cytosol (Figure S11).These findings indicate that AvrLr15 was able to inhibit BAXinduced cell death, suggesting that AvrLr15 has the virulence function in planta.
To date, over 80 Lr resistance genes conferring race-specific seedling or adult plant resistance have been named and deployed in wheat against leaf rust (Kuldeep et al., 2022).Lr15, as a seedling resistance gene, confers resistance against leaf rust at the seedling stage.With the increased deployment of Lr15 in the field, the emergence of new Lr15-breaking races has been reported in many wheat-growing regions worldwide (Dholakia et al., 2013).
To understand the mutation mechanism of AvrLr15, we ampli- at residues P80, M96 P106 compared to AvrLr15 protein sequences (Figure S12), implying that the virulence allele of AvrLr15 is highly conserved.To determine whether this virulent avrLr15 variant could avoid the recognition by Lr15, we cloned the virulent avrLr15 for protein production, and the purified protein was used for infiltration into TcLr15 leaves (Figure 4a). Figure 4b shows that avrLr15 was compromised in inducing cell death in the infiltrated region, similar to the negative controls (buffer and glutathione S-transferase).Next, we examined the localization of avrLr15 and its ability to inhibit BAX-induced cell death.ΔSP avrLr15 was cloned and ligated to the pCamA vector (Figure S13).GFP-tagged avrLr15 localized to the plant nucleus and cytosol, similar to the localization of AvrLr15, after agroinfiltration in N. benthamiana leaves (Figure S14). Figure S15 shows that ΔSP avrLr15 completely suppressed BAX-induced cell death in N. benthamiana, suggesting the virulence allele of AvLr15 is also required for Pt virulence.

To clarify which amino acid change affects the recognition of
AvrLr15 by Lr15, we constructed four mutants with single amino acid changes (AvrLr15 P80A , AvrLr15 ΔP92 , AvrLr15 M96L , AvrLr15 P106S ), and two mutants with double amino acid changes of AvrLr15 (AvrLr15 P80A+ΔP92 , AvrLr15 M96L+P106S ) (Figure S16).The purified mutated proteins were used for infiltration into TcLr15 leaves; avrLr15 was used as a control.The results showed that all the mutated proteins could cause a hypersensitive response (HR) in the leaves of TcLr15, but not in Thatcher wheat lines (Figure 4c (Salcedo et al., 2017).Functional analyses showed that one residue substitution, Q121K, in the coding sequence of Pgt AvrSr50 gene was sufficient to escape recognition by Sr50 (Chen et al., 2017).In addition, the deletion of the AvrSr27 locus in the field Sr27-breaking Pgt clonal lineages has been reported (Upadhyaya et al., 2021).In contrast to amino acid differences in the mature protein, the variant avrSr27-3, without any sequence changes compared to AvrSr27-2, was expressed approximately four-fold lower than the avrSr27-2 variant, and also led to the lack of an avirulence phenotype.Huang et al. (2019) found that the virulent race of Phytophthora sojae does contain the avirulence gene Avr3C-1, but transcription of Avr3C-1 could not be detected by RT-PCR in the virulent race.
Similarly, we found one Lr15-breaking race (64-L-3) does contain the AvrLr15 sequence in the genomes of leaf rust isolates from Australia (Wu et al., 2017) (Figure S17).We hypothesized that the resistancebreaking might be caused by the lack of expression of AvrLr15, so it is necessary to look for the expression of AvrLr15 in virulent races in the future.In addition, extensive nuclear exchange events might occur in natural populations of Pt to generate new gene combinations and pathotypes (Sperschneider et al., 2023).In the current study, we identified Pt avirulence gene AvrLr15 and clarified that mutations in four amino acids in the AvrLr15 protein lead to the loss of avirulence, which will help develop a molecular marker to monitor Pt races in the field for early detection of Lr15-breaking races.AvrLr15 could also be used to confirm the expression of the functional Lr15 protein in F I G U R E 3 AvrLr15 contains a functional signal peptide and can suppress BAX-induced cell death in Nicotiana benthamiana.(a) Functional validation of the putative N-terminal signal peptide of AvrLr15 using the yeast invertase secretion assay.The predicted signal peptide coding sequence of AvrLr15 (SP AvrLr15 ) was cloned into the yeast secretion trap vector pSUC2.The signal peptide of Ps87 was used as a positive control, and empty vector and the signal peptide of Mg87 were used as a negative control.Invertase activity was detected with 2,3,5-triphenyltetrazolium chloride (TTC).(b) Inhibition of BAX-induced cell death on N. benthamiana leaves.Agrobacterium tumefaciens carrying ΔSP AvrLr15 was infiltrated into N. benthamiana leaves, followed 24 h later by infiltration with A. tumefaciens carrying the BAX gene.pCamA and buffer served as a negative control.The same leaf was examined before (top) and after (bottom) being decolourized with the destaining solution.
PTTG_27353 induced an Lr15-dependent cell death response in the wheat TcLr15 line, implying that PTTG_27353 is an AvrLr15 candidate (GenBank accession number: OQ458736.1)that can trigger an Lr15-dependent immune response in wheat leaves.Proteinmediated phenotyping assay in wheat is emerging as a rapid and effective method that facilitates the functional studies of effectors F I G U R E 1 PTTG_27353 effector protein induces Lr15-dependent cell death in the leaves of wheat.(a) The expression profiles of PTTG_27353 during Puccinia triticina infection of the susceptible wheat cultivar Thatcher at different time points.The relative expression of PTTG_27535 was quantified using reverse transcription-quantitative PCR.The y-axis indicates the amounts of PTTG_27535 gene transcript normalized to the β-actin gene.The x-axis indicates sampling times (hours post-inoculation).Values are means ± SEM of three independent experiments.The statistical significance of differences was calculated using a one-way analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) with p < 0.05.For each treatment, three independent biological replicates were used for analysis.(b) Amino acid and nucleotide sequences of PTTG_27353.The red box indicates the signal peptide sequence of PTTG_27353; the cysteine residues are marked in red.(c) SDS-PAGE analysis showed the IPTG-induced and purified PTTG_27535 protein.Lane 1: crude protein of PTTG_27353 induced by IPTG.Lane 2: purified protein PTTG_27353.M: protein marker.(d) Infiltration of PTTG_27535 protein into the leaves of TcLr15 and Thatcher wheat lines.Buffer and purified glutathione S-transferase (GST) protein were used as a negative control.
fied the AvrLr15 genes in 25 natural Pt isolates.PCR amplification products were obtained from these tested isolates and were sequenced.Ten AvrLr15 gene sequences amplified from Lr15breaking isolates all contained nucleotide deletions that led to one amino acid deletion located at amino acid residue P92 and multiple nucleotide changes that resulted in three amino acid substitutions F I G U R E 2 Silencing of AvrLr15 increases virulence of Puccinia triticina.(a) Photobleaching phenotypes were evident on the fourth leaves of plants inoculated by BSMV:PDS at 10 days post-inoculation (dpi).(b) The leaves inoculated with empty vector BSMV:γ used as the control.(c) The phenotype of wheat leaves inoculated with avirulent pathotype PHNT and infected with BSMV:γ.(d) Disease phenotypes of the fourth leaves were pre-inoculated with BSMV:AvrLr15 and infected with the avirulent pathotype PHNT at 10 dpi.
), indicating that the loss of avirulence in AvrLr15 necessitates the concurrent occurrence of four amino acid mutations.The high conservation of avrLr15 variants identified from the Lr15-breaking Pt races suggested natural and direct selection from the Lr15 gene acted on AvrLr15 genes in a specific arms race.Gain of virulence through insertion and deletions has recently been observed in wheat stem rust effector proteins.A microtransposon (mite) insertion in the sixth exon of wheat stem rust fungus Puccinia graminis f. sp.tritici (Pgt) AvrSr35 resulted in early termination of the protein translation, and which thus overcomes the recognition by the Sr35 immune receptor protein